You will need:
Alkaline loading buffer (300mM NaOH, 6mM EDTA, 18% Ficoll 400, 0.15% bromocresol green, 0.25% xylene cyanol FF)
10 x Alkaline agarose electrophoresis buffer (500mM NaOH, 10mM EDTA, pH 8.0, made fresh for each use)
1) Melt the required amount of powdered agarose in a measured quantity of water, allow to cool to approximately 60°C and add 0.1 volumes 10 x alkaline agarose electrophoresis buffer. Pour gels as described in 'Standard Agarose Electrophoresis'.
N.B: Agarose should not be melted in the presence of NaOH as this causes the hydrolysis of the polysaccharides present in the gel.
2) Samples to be analysed (usually 1-3ul) should be added to 10-20ul 1 x akaline agarose electrophoresis buffer and to this should be added 0.2 volumes of alkaline loading buffer.
3) Load samples and carry out electrophoresis at a maximum of 5V/cm until the bromocresol green has migrated approximately two thirds of the length of the gel.
N.B: It is imperative to prevent the gel and buffer temperature getting above 40-45°C as alkaline hydrolysis of the agarose polysaccharides will occur causing dissolution of the gel. The voltage gradient should therefore be adjusted accordingly. Only gel rigs with a large buffer volume (and therefore a large heat sink) or a cooling system should be run at 5V/cm.
If the samples contain 32P-labelled DNA, after electrophoresis the gels can fixed for 30 minutes in 7% TCA(w/v), dried and subjected to autoradiography.
Dr. Simon Dawson,
Department of Biochemistry,
University of Nottingham,
The Medical School ,
Clifton Boulevard ,
Tel: +44 (0)115 9249924 ex.44787,
FAX: +44 (0)115 9422225,
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